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Molecular mechanisms underlying the corneal endothelial pump
Experimental Eye Research 95 (2012) 2e7
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Molecular mechanisms underlying the corneal endothelial pump Joseph A. Bonanno* Indiana University, School of Optometry, 800 E Atwater Avenue, Bloomington, IN 47405, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 March 2011 Accepted in revised form 7 June 2011 Available online 15 June 2011
The corneal endothelium is responsible for maintaining the hydration of the cornea. This is through a “Pump-Leak” mechanism where the active transport properties of the endothelium represent the “Pump” and the stromal swelling pressure represents the “Leak”. For the “Pump”, Naþ, Kþ ATPase activity and the presence of HCO3, Cl, and carbonic anhydrase activity are required. Several basolateral (stromal side) anion transporters, apical (facing the aqueous humor) ion channels and water channels have been identified that could support a model for ion secretion as the basis for the endothelial pump, however evidence of sustained anion fluxes, osmotic gradients or the need for water channels is lacking. This has prompted consideration of other models, such as Electro-osmosis, and consideration of metabolite flux as components of the endothelial pump. Although the conditions under which the “Pump” is supported are known, a complete model of the endothelial “Pump” has yet to emerge. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: corneal endothelium transparency ion & fluid transport
1. Introduction The topic of molecular mechanisms underlying the corneal endothelial pump was extensively reviewed in 2003 (Bonanno, 2003). The purpose here is to summarize the important features of the endothelial pump and provide an update on recent significant findings. Development of new techniques, e.g. siRNA knockdown and in vivo viral transfection of shRNA, has produced more specific testing of pump features. Similarly, the use of knockout models (e.g., AQP1) has prompted significant re-thinking of the nature of solutefluid coupling. These molecular approaches together with new studies using customary pharmacological approaches for studying corneal endothelial fluid transport have led to re-evaluations of the corneal endothelial pump. 2. Background The cornea is the major refractive element of the eye. This requires optical transparency and smooth curved surfaces. The cornea has five layers: the outer epithelium, Bowman’s (basement) membrane, the stroma, Descemet’s (basement) membrane, and the inner surface endothelium. The smooth regular surface, tight packing of cells, relative paucity of organelles (especially mitochondria), and lack of blood vessels in the stratified squamous corneal epithelium (w50 mm) reduce light scatter thereby contributing to transparency. The corneal
* Tel.: þ1 812 855 4440; fax: þ1 812 855 7045. E-mail address: [email protected]. 0014-4835/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.exer.2011.06.004
stroma reduces light scatter by its regular spacing among collagen fibers and uniform diameter of fibers. This contrasts with the sclera where spacing and fiber diameters have a very broad distribution. The regular spacing of the stromal fibers is controlled by the hydrophilic glycosaminoglycan (GAGs) ground substance. When the hydration of the stroma is w3.5 mg H2O/mg dry tissue or less, the stroma is relatively transparent. A slit-lamp view of the stroma however, indicates that it scatters more light than the epithelium. This is due to the thin flat keratocytes that lie between stromal lamellae. Keratocytes are responsible for producing collagen and GAGs (among many other extracellular substances) and will be activated during trauma to repair the stroma. Repair alters the expression profile of keratocytes, which can lead to increased light scatter. In contrast, the corneal endothelium is a thin (4 mm) confluent monolayer that has a very high density of mitochondria, but because of its extremely short optical path length, scatters very little light. The epithelium provides the barrier to the outside world, the stroma provides the refractive shape and the endothelium maintains the nutrition of the corneal cells and the hydration of the stroma. Except for oxygen, all the nutrients for the cornea come from the aqueous humor and through the endothelium. For example, glucose transporters are present on both the apical (aqueous humor side) and basolateral (stromal side) endothelial cell membranes to allow transcellular glucose flux (Kumagai et al., 1994). Moreover, 85% of the glucose consumed by the cornea is converted to lactate, which diffuses posteriorly across the endothelium (Riley, 1969). The corneal endothelium also maintains the hydration of the stroma through active transport mechanisms. Because of the swelling pressure (w60 mmHg) exerted by molecular
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repulsion from the highly negatively charged stromal GAGs, bare stroma can swell to many times its normal thickness (see (Kwok and Klyce, 1990) for further details) and as a consequence the spacing between fibers becomes non-uniform, light scatter increases, and corneal transparency is lost. This tendency to swell is counteracted by the endothelial pump through the membrane active transport mechanisms. Steady-state hydration occurs when the endothelial pump rate equals the GAG driven leak. Maurice (Maurice, 1981) called this the “Pump-Leak” mechanism for maintenance of corneal hydration and transparency. Because of the presence of the continuous leak, loss of endothelial ion transport activity leads to corneal edema, loss of transparency, and impaired vision. 3. Corneal endothelial pump description The endothelial pump function is best demonstrated using rabbit corneas mounted in modified Ussing chambers in vitro. If carefully mounted and perfused with appropriate media, the cornea will maintain its thickness for several hours. Another approach is to remove the epithelium, expose the anterior stroma for a short period to Ringer’s solution allowing the stroma to swell, remove the anterior solution and replace it with silicone oil. The corneal thickness will then slowly decrease exponentially in a process called deturgescence. From these experiments it was determined that the cornea will swell, or not deturgesce, if the endothelium is exposed to the cardiac glycoside ouabain, an inhibitor of the Naþ, Kþ ATPase, indicating that the endothelial pump is dependent on primary active transport. Removal of HCO3 from the endothelial perfusing solution had a similar inhibitory effect on the pump and addition of carbonic anhydrase inhibitors slowed the pump by w30%, indicating a major role for HCO3 (Dikstein and Maurice, 1972; Fischbarg and Lim, 1974; Hodson and Miller, 1976; Riley et al., 1995). Because Cl removal did not cause initial swelling, the early pump model was described as an exclusiveHCO3 secretory mechanism. Subsequent studies indicated that Cl was important for maintaining the pump (Winkler et al., 1992), suggesting that an anion exchanger, (Cl/HCO3) played a role. Consistent with an anion transport mechanism, anion transport blockers, e.g. DIDS (4,40 -Diisothiocyano-2,20 -stilbenedisulfonic acid), significantly slow the pump(Kuang et al., 2004a; Riley et al., 1996). There must also be a role for secondary Naþ fluxes because amiloride, which blocks the Naþ/Hþ exchanger and at high concentrations the Naþ channel ENaC (Epithelial Sodium Channel), also slows fluid transport (Liebovitch and Fischbarg, 1982). Assembling these disparate observations into a clear model for endothelial pump function has been very challenging and is described below.
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Moreover, the basolateral membrane HCO3 permeability of the corneal endothelium is significantly higher than the apical permeability (Bonanno et al., 1999) and with the exception of apical anion channels; no apical HCO3 transporter has been identified. Fig. 1 shows a possible bicarbonate transport model for the corneal endothelium. The basolateral Naþ, Kþ ATPase creates a low intracellular [Naþ] and high intracellular [Kþ], and in conjunction with Kþ channels a negative membrane potential of about 55 mV (Watsky and Rae, 1991). On the basolateral side there are two bicarbonate transporters, a 1Naþ:2HCO3 cotransporter (SLC4A4,NBCe1; Sodium Bicarbonate Cotransporter electrogenic) and the Cl/HCO3 exchanger (SLC4A2, AE2; Anion Exchanger),as well as a Naþ/Hþ exchanger (SLC9A6, NHE1; Sodium Hydrogen Exchanger). NBCe1 can directly load HCO3 into the cell. The Naþ/Hþ exchanger can indirectly load HCO3 into the cell because removal of protons favors formation of HCO3 from CO2, catalyzed by carbonic anhydrase II. Conversely, the Cl/HCO3 exchanger is poised to remove HCO3 from the cell and add Cl. Moreover, a basolateral Naþ:Kþ:2Cl cotransporter helps load intracellular chloride to w40 mM, above electrochemical equilibrium (w12 mM). On the apical side, at least two anion channels have been identified: CFTR (Cystic Fibrosis Transmembrane conductance Regulator) and CaCC (Calcium-activated Chloride Channel). Anion permeability of both channels favors Cl vs HCO3 by w4:1.There is no evidence for apical Cl/HCO3 exchange. As such the model (see Fig. 1) predicts that HCO3 is taken up on the basolateral side and efflux of HCO3 across the apical side is through anion channels driven by the negative membrane potential. Fig. 1 shows an additional potential indirect route for apical HCO3 efflux. Because the influx mechanisms for HCO3 exceed direct efflux mechanisms and the differential must be converted to CO2 (Bonanno and Giasson, 1992), the CO2 can diffuse in any direction and some will diffuse across the apical membrane where carbonic anhydrase IV could catalyze the conversion back to
4. The anion (bicarbonate & chloride) secretion model Fluid secretion that is coupled to ion fluxes is dependent on active transport mechanisms to produce local osmotic gradients that move water across the cellular layer. This is best described for secretory glands and kidney reabsorption processes. More recently, evidence for direct coupling of water to ion fluxes in cotransporters has also been presented (Hamann et al., 2003; Meinild et al., 2000) and could have a significant role in fluid absorption across the intestinal mucosa (Loo et al., 1996). The best tissue models for bicarbonate secretion are the gall bladder and pancreas. In both tissues transporters and anion channels have been identified in both basolateral and apical membranes that could provide the net transcellular bicarbonate flux that can unequivocally be measured. In contrast, several groups have measured HCO3 and Cl fluxes across the rabbit corneal endothelium. However, there is no consensus that a net anion flux can be generated (Bonanno, 2003).
Fig. 1. Bicarbonate Secretion Model for Endothelial Pump. Fluid coupled anion secretion requires transendothelial net flux of Cl and/or HCO3. The movement of net negative charge creates a small potential difference (0.5 mV, apical side negative) that attracts Naþ through the paracellular pathway & across the tight junction (TJ). The net flux of NaHCO3 and/or NaCl constitutes the osmotic driving force for water movement. HCO3 uptake on the basolateral membrane is through the actions of the 1Naþ:2HCO3 cotransporter (NBCe1) and the Naþ/Hþ exchanger (NHE1). Cl uptake is primarily via the Naþ:Kþ:2Clcotransporter (NKCC1) and the Cl/HCO3 exchanger (AE2). The high intracellular [Cl] and [HCO3], together with the negative membrane potential can then drive anions across the apical membrane through anion selective channels. An additional route for net HCO3 flux is for the high intracellular [HCO3] to be converted to CO2, facilitated by carbonic anhydrase II (CAII), apical CO2 diffusion and conversion back to HCO3, facilitated by carbonic anhydrase IV (CAIV) at the apical surface. This pathway is less attractive because it does not contribute to the transendothelial potential.
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HCO3 and thereby contribute to a local buildup of osmotic pressure at the apical membrane. The following sections discuss recent studies of several corneal endothelial transport mechanisms and their potential contribution to an anion secretion model. 4.1. NKCC1 The role for Cl is unclear. Intracellular [Cl] is about 40 mM in bovine endothelium (Srinivas et al., 1998), which is above electrochemical equilibrium. This is due to the presence of a bumetanide sensitive basolateral Naþ:Kþ:2Cl cotransporter (NKCC1) (Diecke et al., 1998; Diecke et al., 2005; Jelamskii et al., 2000). Together with the apical Cl channels, the potential for Cl secretion exists. One study could not show any effect of the Naþ:Kþ:2Cl blocker bumetanide on rabbit corneal deturgescence (Riley et al., 1997), however small, but significant effects were noted in a later study (Diecke et al., 2005). Certainly NKCC1 is involved in regulation of cell volume, however given the lack of clear evidence for a net transendothelial Cl flux, the role NKCC1 could have in the endothelial pump is uncertain. One suggestion is that Cl efflux through anion channels offsets the membrane hyperpolarizing effect of the electrogenic1Naþ:2HCO3 cotransporter. By keeping the membrane potential relatively depolarized through Cl efflux, the driving force for continual 1Naþ:2HCO3 influx is maintained. 4.2. NBCe1 The dependence of endothelial fluid transport on active transport and the presence of bicarbonate suggest an important role for the 1Naþ:2HCO3 cotransporter (NBCe1). NBCe1 has two splice variants, pNBCe1 and kNBCe1, for pancreas and kidney type, respectively. The kidney proximal tubule form of NBC (kNBC) has a 1:3 stoichiometry, which favors cellular efflux of HCO3, and the pancreas form of NBC (pNBC) has a 1:2 stoichiometry, which favors HCO3 influx. However, more recent studies have shown that the stoichiometry of either kNBC or pNBC can change depending on the cell type in which it is expressed (Gross et al., 2001). Previous reports indicated the pNBC variant was expressed in bovine (Sun et al., 2000), human (Sun and Bonanno, 2003)and rat corneal endothelium(Bok et al., 2001), and one report suggested both variants were expressed in human endothelium (Usui et al., 1999).Immunohistochemistry studies in cultured and fresh bovine, rat, and human endothelium indicate that NBCe1 exclusively locates to the basolateral membrane; however, one report (Diecke et al., 2004) suggests apical expression as well. NBCe1 specific siRNA knockdown reduced NBCe1 expression by 80e90% four days post transfection in cultured bovine corneal endothelial cells (Li et al., 2005). This treatment significantly reduced basolateral HCO3 permeability and also reduced the basolateral to apical HCO3 flux induced by basolateral to apical [HCO3] gradient. Importantly, apical HCO3 permeability and apical to basolateral HCO3 flux was unaffected by siRNA knockdown. This study shows that functional NBCe1 is only present on the basolateral membrane and that it is necessary for a large portion of basolateral HCO3 influx. 4.3. Calcium-activated chloride channels In bovine corneal endothelial cells, apical HCO3 permeability can be enhanced by increasing [Ca2þ]i via either activation of purinergic receptors (e.g., by ATP) or inhibition of the sarcoendoplasmic reticulum Ca2þ-ATPase (SERCA) (Xie et al., 2002). In either case there is a release of Ca2þ from intracellular stores with concomitant Ca2þ entry from the extracellular bath through storeoperated channels in a process called capacitive calcium entry (CCE). CCE agonists enhanced apical HCO3 permeability in Ca2þrich Ringer solution, but not in the absence of extracellular Ca2þ,
which was blocked by the CCE inhibitor2-aminoethoxydiphenyl borate (2-APB) (Zhang et al., 2006; Zhang et al., 2002). ATP release from endothelial cells does occur and can be stimulated by injury or cell volume increase (Srinivas et al., 2001) indicating that this pathway could be important during cellular stress. A likely candidate for the apical Ca2þsensitive channel is CLCA (chloride channel, calcium-activated) (Cunningham et al., 1995). CLCA1 antiserum detected the 90kD band in corneal endothelium. Immunofluorescence staining showed an apical membrane location (Zhang et al., 2006). In endothelial cells transfected with CLCA1 specific siRNA, CLCA1 expression was reduced by 80%. Activating CCE increased apical HCO3 permeability in siControl transfected cells, while having no effect in CLCA1 specific siRNA transfected cells. Baseline HCO3 permeability however, was not different between control and siRNA treated cells. These studies indicate that the calcium-activated chloride channel (CLCA1) is expressed in corneal endothelial cells and can contribute to Ca2þ dependent apical HCO3 permeability, but not resting permeability across the corneal endothelium. Since release of ATP activates the purinergic pathway, this channel may have a role during stress, e.g. inflammation, to enhance “Pump” activity. 4.4. CFTR Increasing intracellular [cAMP] by activating membrane adenylyl cyclases with forskolin stimulates an NPPB (5-nitro-2-(3phenylpropylamin benzoic acid)) and glibenclamide-inhibitable apical Cl and HCO3 permeability that has been identified as CFTR (Cystic Fibrosis Transmembrane conductance Regulator) in bovine corneal endothelium (Sun and Bonanno, 2002). Knocking down CFTR expression using siRNA eliminated forskolin induced increases in apical Cl and HCO3 permeability (Li et al., 2008). However, the siRNA knockdown had no effect on baseline unstimulated anion permeability. Stimulation of CFTR can also induce a steady-state apical bath alkalinization. In CFTR siRNA treated cells, the baseline bath pH was similar to control, but stimulation did not produce an apical alkalinization. These findings indicate that cAMP induced increases in apical HCO3 permeability in bovine corneal endothelium are due to CFTR. However, CFTR does not have a major role in determining baseline apical chloride or HCO3 permeability. This is consistent with reports indicating that there are no corneal abnormalities in humans with cystic fibrosis and studies showing that a specific CFTR blocker had no significant effect on baseline corneal thickness (Diecke et al., 2004). CFTR may, like CLCA, have a role only during stimulated stressful conditions. 4.5. CA IV As described in Fig. 1, there is the potential for apical CO2 flux contributing to net basolateral to apical HCO3 flux. This requires an apical carbonic anhydrase. An apical CAIV is present in bovine corneal endothelium (Sun et al., 2008). The relatively membrane impermeant carbonic anhydrase inhibitor benzolamide or CAIV siRNA knockdown reduced apical CO2 fluxes due to an imposed [CO2] gradient by w20%, however there was no effect on HCO3 permeability or HCO3 flux (Sun et al., 2008). If a significant net flux of CO2 occurred across the apical membrane with conversion to HCO3 þ Hþ (Fig. 1), then blocking the apical CAIV would cause an alkalinization of the apical bath. However, apically applied benzolamide and siRNA treated cells showed an acidification of the apical side relative to basolateral, which is inconsistent with a net cell to apical CO2 flux. Because CAIV does not facilitate steady-state cell to apical CO2 flux, apical HCO3 permeability, or basolateral to apical HCO3 flux, but apparently maintains apical side pH, CAIV may have a role in buffering the apical surface.
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4.6. Role of NBCe1: in vivo studies
6. Aquaporins
A mouse knockout for NBCe1 exists, however NBCe1 knockouts have significant developmental and systemic abnormalities and die shortly after birth, limiting their usefulness in analyzing the role of NBCe1 in the cornea. Therefore, to follow up on the previous in vitro studies, lentiviral vectors encoding NBCe1 specific shRNA were constructed and injected into the anterior chamber of live rabbits to determine if the bicarbonate fluxes produced by NBCe1 were needed to support corneal endothelial fluid transport in vivo (Liu et al., 2010). This study showed that significant (up to 90%) endothelial NBCe1 knockdown could occur in vivo, however there was large variability in knockdown efficiency. If transendothelial HCO3 transport were an integral part of the endothelial pump, then knocking down NBCe1 should induce significant corneal swelling. Surprisingly, there was no effect on corneal thickness. However, relative to control eyes that were injected with a Lentivirus encoding a scrambled sequence shRNA, the eyes receiving the NBCe1 specific shRNA were more sensitive to the topical carbonic anhydrase inhibitor brinzolamide, i.e. there was more corneal swelling. These results were interpreted to suggest that the corneal endothelium has a large functional reserve (e.g., NBCe1 & CA activity), however they could also be interpreted to suggest that transendothelial HCO3 flux is not an integral part of the “Pump”. In summary, the recent studies presented above do not directly support the anion secretion model.
The role of aquaporins in fluid transport has been controversial. Knockout models of AQP1, the most ubiquitous aquaporin, showed essentially no phenotype unless the animal was stressed by withholding water (Agre, 1998). Subsequent studies indicated that knockout of aquaporins had effects on secretory tissues if the rate of secretion was relatively high (Ma et al., 2000; Ma et al., 1999). Corneal endothelium has a high density of AQP1 located on both apical and basolateral membranes (Kuang et al., 2004b). In the knockout mouse, corneal thickness is thinner than wild type rather than the expected thicker more hydrated cornea if AQP1 was an important part of the “Pump”. The endothelial osmotic permeability is significantly reduced, as expected, and the rate of corneal thickness change in response to an osmotic challenge is slowed. These results suggest that AQP1 is present as a passive water pathway that speeds stromal hydration changes due to osmotic pressure fluctuations. In normal physiological situations this would include eye closure, which swells the cornea by w4%, or exposure to hypo-osmotic conditions, e.g. swimming. This is consistent with the findings in other tissues that aquaporins do not have a role in secretion if the rate is relatively low.
5. The electro-osmosis model The conventional view of epithelial cell secretion and absorption of water is that epithelial cell layers create local osmotic differences in the lateral spaces between the cells and/or on the apical surfaces of cells within an unstirred layer. The osmotic gradients generated serve as the driving forces for water movement across the epithelium. However, in many epithelial cells, including the corneal endothelium, there is no evidence that these gradients exist. This has led to the consideration of other mechanisms, such as ElectroOsmosis (Sanchez et al., 2002). In this process, cells generate a transepithelial potential (0.5 mV apical side negative in corneal endothelium), through the concerted action of apical anion channels (HCO3 and Cl), that draws counter-ions, i.e. Naþ, through a paracellular pathway (the lateral space between cells) lined with fixed negative charges that make the paracellular pathway Naþ specific. This produces electro-osmotic fluid coupling across the tight junction. Evidence for electro-osmosis and against other modes of fluid transport is recently reviewed (Fischbarg, 2010). For electroosmosis, a “leaky” junction with low specific resistance (20 U cm2) must have a relatively intense current density. This high Naþ current generates fluid movement by electro-osmotic coupling. The Naþ current loop is complete due to the presence of apical Naþ channels (ENaC) (Mirshahi et al., 1999). Of interest, apical membrane density of ENaC can be enhanced by SGK1 activity (serum glucocorticoid kinase), which has also been shown to increase Na/K-ATPase activity in corneal endothelium (Hatou et al., 2009; Rauz et al., 2003). This could be interpreted in favor of electro-osmosis or the increased ENaC density may simply be needed to offset the hyperpolarizing effect of increased Na/K-ATPase activity in order to maintain a stable membrane potential. Moreover, glucocorticoids thin rabbit (Hara, 1970), but not human corneas (Baum and Levene, 1968). A major problem with electro-osmosis in corneal endothelium is that a significant reduction in endothelial cell density, which is a normal process of ageing, does not result in reductions in endothelial function or production of corneal edema. The integrated effect of the high current density would be dissipated since the total paracellular area is significantly decreasing with age.
7. Fluid transport agonists The most highly studied fluid transport agonists in corneal endothelium are those that result in increased [cAMP]. Adenosine was the first agonist discovered (Dikstein and Maurice, 1972) and was subsequently shown that it increased [cAMP] in endothelial cells (Riley et al., 1996) through activation of A2b receptors (TanAllen et al., 2005). Other agonists that increase [cAMP], e.g. forskolin or the phosphodiester inhibitor rolipram, also cause corneal thinning (Wigham et al., 2000). It is important to note that this could occur in de-epithelialized preparations so it was not due to the well-characterized corneal epithelial cAMP dependent Cl secretion (Klyce, 1975). Besides being protective for corneal endothelial cells (Li et al., 2011), increased cAMP will phosphorylate and activate the apical CFTR channel leading to increased Cl and HCO3 flux (Sun et al., 2003). It also relaxes endothelial cells leading to better cellecell apposition and increased transendothelial resistance (Ramachandran and Srinivas, 2010). ATP is also a potential fluid transport agonist. Purinergic receptor activation increased apical anion channel permeability as described above. Moreover, ATP released by the endothelial cells, can be broken down to adenosine by the presence of eco ATPases (Soltau et al., 1993). Again, the presence of this physiologic response is probably in place to stimulate endothelial function during inflammatory, oxidative, or traumatic stress to the cell layer, but this will need to be tested. 8. Facilitated lactate transport Often overlooked is the fact that in the in vivo cornea there are substantial bulk gradients of ions, e.g. glucose, HCO3 and lactate (Chhabra et al., 2009). [HCO3] is higher in the anterior chamber than in the cornea, especially when the eyelid is open. Thus HCO3 will diffuse into the cornea passively in the opposite direction of the putative bicarbonate secretory mechanism. The cornea gets all of its glucose from the anterior chamber. Glucose in the cornea is consumed thereby maintaining an inward gradient for glucose diffusion. The high concentration of mitochondria in the endothelial cells would minimize glucose consumption allowing for supply to the very glycolytic keratocytes and epithelial cells. Overall, Riley (1969) has shown that 85% of glucose consumed in the cornea is converted to lactic acid. Therefore, for every 100 glucose molecules
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channel activation and greater barrier function. Further studies are needed to determine if this mechanism could be used as a therapy for corneal edema. References
Fig. 2. Facilitated Lactate Transport Model. The bulk movement of lactic acid from cornea to anterior chamber via transcellular Monocarboxylate Cotransporters (MCTs) could contribute to net fluid movement out of the cornea. Intracellular buffering by the high intracellular [HCO3], provided by the 1Naþ:2HCO3 (NBCe1), Naþ/Hþ exchanger (NHE1), and carbonic anhydrase II work to facilitate basolateral lactate:Hþ entry. Apical efflux through an MCT is facilitated by robust apical surface buffering, which is supplied by aqueous humor HCO3 and apical surface carbonic anhydrase IV.
entering the cornea, 170 lactate molecules are produced. Klyce (1981) demonstrated that the epithelium is impermeable to lactate and that the [lactate] in the cornea was about twice that in the aqueous humor, so there is a large gradient for lactate flux from cornea (w13 mM) to anterior chamber (w7 mM) across the endothelium. Lactate:Hþ cotransport was demonstrated in the endothelium (Giasson and Bonanno, 1994) and the monocarboxylate transporters (MCTs) responsible are being studied in our laboratory. Corneal hypoxia increases [lactate] and causes corneal swelling (Klyce, 1981) indicating that lactate is an important osmolyte. It follows that any process that facilitates lactate efflux from the cornea will help maintain dehydration. Conversely, any interference with lactate efflux will cause corneal swelling. Fig. 2 illustrates a model for lactate efflux across the endothelium that is facilitated by HCO3 buffering, carbonic anhydrase activity, 1Naþ:2HCO3 cotransport, and Naþ/Hþ exchange. An important role for buffering acid is supported by studies indicating that deturgescence can be maintained in the absence of HCO3 if a high concentration of other buffers are present (Doughty and Maurice, 1988). Studies in our laboratory are currently examining the extent to which this process may contribute to the endothelial pump. 9. Conclusion The corneal endothelial pump is an active transport dependent process that requires both Cl and HCO3, but is highly dependent on HCO3 and facilitated by carbonic anhydrase activity. While a HCO3 secretory model for the pump has been preferred, there are several issues that argue against this interpretation. These include: the lack of apical anion transporters, equivocal measures of net anion fluxes from stroma to anterior chamber (probably due to the extremely leaky nature of the endothelium), no data indicating the presence of local osmotic gradients, a bulk fluid HCO3 gradient that is against the putative secretory mechanism, and the greater potential for a Cl secretory mechanism. Alternatives such as electro-osmosis are being considered, but this suffers from the fact that significant reductions in electro-osmotic potential, i.e. the paracellular space, do not have direct effects on fluid pumping. The lactate efflux mechanism is attractive, but requires significant experimental testing. Increasing endothelial cAMP enhances pump function by a combination of anion
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Gross, E., Hawkins, K., Abuladze, N., Pushkin, A., Cotton, C.U., Hopfer, U., Kurtz, I., 2001. The stoichiometry of the electrogenic sodium bicarbonate cotransporter NBC1 is cell-type dependent. J. Physiol. 531 (Pt 3), 597e603. Hamann, S., Kiilgaard, J.F., la Cour, M., Prause, J.U., Zeuthen, T., 2003. Cotransport of Hþ, lactate, and H2O in porcine retinal pigment epithelial cells. Exp. Eye Res. 76 (4), 493e504. Hara, T., 1970. Effect of topical application of hydrocortisone on the corneal thickness. Exp. Eye Res. 10 (2), 302e312. Hatou, S., Yamada, M., Mochizuki, H., Shiraishi, A., Joko, T., Nishida, T., 2009. The effects of dexamethasone on the Na, K-ATPase activity and pump function of corneal endothelial cells. Curr. Eye Res. 34 (5), 347e354. Hodson, S., Miller, F., 1976. The bicarbonate ion pump in the endothelium which regulates the hydration of rabbit cornea. J. Physiol. 263, 563e577. Jelamskii, S., Sun, X.C., Herse, P., Bonanno, J.A., 2000. Basolateral Naþ-Kþ-2Clcotransport in cultured and fresh bovine corneal endothelium. Invest. Ophthalmol. Vis. Sci. 41, 488e495. Klyce, S., 1975. Transport of Cl, Na, and water by the rabbit corneal epithelium at resting potential. Am. J. Physiol. 228, 1446e1452. Klyce, S., 1981. Stromal lactate accumulation can account for corneal oedema osmotically following epithelial hypoxia in the rabbit. J. Physiol. (Lond.) 321, 49e64. Kuang, K., Li, Y., Yiming, M., Sanchez, J.M., Iserovich, P., Cragoe, E.J., Diecke, F.P., Fischbarg, J., 2004a. Intracellular [Naþ], Naþ pathways, and fluid transport in cultured bovine corneal endothelial cells. Exp. Eye Res. 79 (1), 93e103. Kuang, K., Yiming, M., Wen, Q., Li, Y., Ma, L., Iserovich, P., Verkman, A.S., Fischbarg, J., 2004b. Fluid transport across cultured layers of corneal endothelium from aquaporin-1 null mice. Exp. Eye Res. 78 (4), 791e798. Kumagai, A.K., Glasgow, B.J., Pardridge, W.M., 1994. GLUT1 glucose transporter expression in the diabetic and nondiabetic human eye. Invest. Ophthalmol. Vis. Sci. 35 (6), 2887e2894. Kwok, L.S., Klyce, S.D., 1990. Theoretical basis for an anomalous temperature coefficient in swelling pressure of rabbit corneal stroma. Biophys. J. 57 (3), 657e662. Li, J., Allen, K.T., Sun, X.C., Cui, M., Bonanno, J.A., 2008. Dependence of cAMP meditated increases in Cl- and HCO(3)- permeability on CFTR in bovine corneal endothelial cells. Exp. Eye Res. 86 (4), 684e690. Li, J., Sun, X.C., Bonanno, J.A., 2005. Role of NBC1 in apical and basolateral HCO3permeabilities and transendothelial HCO3- fluxes in bovine corneal endothelium. Am. J. Physiol. Cell Physiol. 288 (3), C739eC746.
J.A. Bonanno / Experimental Eye Research 95 (2012) 2e7 Li, S., Allen, K.T., Bonanno, J.A., 2011. Soluble adenylyl cyclase mediates bicarbonatedependent corneal endothelial cell protection. Am. J. Physiol. Cell Physiol. 300 (2), C368eC374. Liebovitch, L., Fischbarg, J., 1982. Effects of inhibitors of passive Naþ and HCO3 fluxes on electrical potential and fluid transport across rabbit corneal endothelium. Curr. Eye Res. 2, 183e186. Liu, C., Cheng, Q., Nguyen, T., Bonanno, J.A., 2010. Knockdown of NBCe1 in vivo compromises the corneal endothelial pump. Invest. Ophthalmol. Vis. Sci. 51 (10), 5190e5197. Loo, D.D., Zeuthen, T., Chandy, G., Wright, E.M., 1996. Cotransport of water by the Naþ/glucose cotransporter. Proc. Natl. Acad. Sci. U S A 93 (23), 13367e13370. Ma, T., Fukuda, N., Song, Y., Matthay, M.A., Verkman, A.S., 2000. Lung fluid transport in aquaporin-5 knockout mice. J. Clin. Invest. 105, 93e100. Ma, T., Song, Y., Gillespie, A., Carlson, E.J., Epstein, C.J., Verkman, A.S., 1999. Defective secretion of saliva in transgenic mice lacking aquaporin-5 water channels. J. Biol. Chem. 274, 20071e20074. Maurice, D., 1981. The Cornea and Sclera. Academic Press, New York. Meinild, A.-K., Loo, D.D.F., Pajor, A.M., Zeuthen, T., Wright, E.M., 2000. Water transport by the renal Naþ-dicarboxylate cotransporter. Am. J. Physiol. Renal Physiol. 278, F777eF783. Mirshahi, M., Nicolas, C., Mirshahi, S., Golestaneh, N., d’Hermies, F., Agarwal, M.K., 1999. Immunochemical analysis of the sodium channel in rodent and human eye. Exp. Eye Res. 69 (1), 21e32. Ramachandran, C., Srinivas, S.P., 2010. Formation and disassembly of adherens and tight junctions in the corneal endothelium: regulation by actomyosin contraction. Invest. Ophthalmol. Vis. Sci. 51 (4), 2139e2148. Rauz, S., Walker, E.A., Murray, P.I., Stewart, P.M., 2003. Expression and distribution of the serum and glucocorticoid regulated kinase and the epithelial sodium channel subunits in the human cornea. Exp. Eye Res. 77 (1), 101e108. Riley, M., 1969. Glucose and oxygen utilization by the rabbit cornea. Exp. Eye Res. 8, 193e200. Riley, M., Winkler, B., Czajkowski, C., Peters, M., 1995. The roles of bicarbonate and CO2 in transendothelial fluid movement and control of corneal thickness. Invest. Ophthalmol. Vis. Sci. 36, 103e112. Riley, M., Winkler, B., Starnes, C., Peters, M., 1996. Adenosine promotes regulation of corneal hydration through cyclic adenosine monophosphate. Invest. Ophthalmol. Vis. Sci. 37, 1e10. Riley, M., Winkler, B., Starnes, C., Peters, M., 1997. Fluid and ion transport in corneal endothelium: insensitivity to modulators of Na-K-2Cl cotransport. Am. J. Physiol. 273, C1480eC1486. Sanchez, J.M., Li, Y., Rubashkin, A., Iserovich, P., Wen, Q., Ruberti, J.W., Smith, R.W., Rittenband, D., Kuang, K., Diecke, F.P., Fischbarg, J., 2002. Evidence for a central role for electro-osmosis in fluid transport by corneal endothelium. J. Membr. Biol. 187 (1), 37e50. Soltau, J.B., Zhou, L.X., McLaughlin, B.J., 1993. Isolation of plasma membrane domains from bovine corneal endothelial cells. Exp. Eye Res. 56 (1), 115e120.
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Srinivas, S.P., Bonanno, J.A., Hughes, B.A., 1998. Assessment of swelling-activated Clchannels using the halide-sensitive fluorescent indicator 6-methoxy-N-(3sulfopropyl)quinolinium. Biophys. J. 75 (1), 115e123. Srinivas, S.P., Mutharasan, R., Sun, X.C., Bonanno, J.A., 2001. Stretch-activated ATP release by corneal endothelium. Invest. Ophthalmol Vis. Sci. 42 (4), S665. Sun, X.C., Bonanno, J.A., 2002. Expression, localization, and functional evaluation of CFTR in bovine corneal endothelial cells. Am. J. Physiol. Cell Physiol. 282 (4), C673eC683. Sun, X.C., Bonanno, J.A., 2003. Identification and cloning of the Na/HCO(3) cotransporter (NBC) in human corneal endothelium. Exp. Eye Res. 77 (3), 287e295. Sun, X.C., Bonanno, J.A., Jelamskii, S., Xie, Q., 2000. Expression and localization of NaHCO3 cotransporter in bovine corneal endothelium. Am. J. Physiol. Cell Physiol. 279, C1648eC1655. Sun, X.C., Li, J., Cui, M., Bonanno, J.A., 2008. Role of carbonic anhydrase IV in corneal endothelial HCO3-transport. Invest. Ophthalmol. Vis. Sci. 49 (3), 1048e1055. Sun, X.C., Zhai, C.B., Cui, M., Chen, Y., Levin, L.R., Buck, J., Bonanno, J.A., 2003. HCO(3)(-)-dependent soluble adenylyl cyclase activates cystic fibrosis transmembrane conductance regulator in corneal endothelium. Am. J. Physiol. Cell Physiol. 284 (5), C1114eC1122. Tan-Allen, K.Y., Sun, X.C., Bonanno, J.A., 2005. Characterization of adenosine receptors in bovine corneal endothelium. Exp. Eye Res. 80 (5), 687e696. Usui, T., Seki, G., Amano, S., Oshika, T., Miyata, K., Kunimi, M., Taniguchi, S., Uwatoko, S., Fujita, T., Araie, M., 1999. Functional and molecular evidence for Na(þ)-HCO3- cotransporter in human corneal endothelial cells. Pflugers Arch. 438, 458e462. Watsky, M.A., Rae, J.L., 1991. Resting voltage measurements of the rabbit corneal endothelium using patch-current clamp techniques. Invest. Ophthalmol. Vis. Sci. 32, 106e111. Wigham, C.G., Turner, H.C., Swan, J., Hodson, S.A., 2000. Modulation of corneal endothelial hydration control mechanisms by Rolipram. Pflugers Arch. 440, 866e870. Winkler, B., Riley, M., Peters, M., Williams, F., 1992. Chloride is required for fluid transport by the rabbit corneal endothelium. Am. J. Physiol. 262, C1167eC1174. Xie, Q., Zhang, Y., Zhai, C., Bonanno, J.A., 2002. Calcium influx factor from cytochrome P-450 metabolism and secretion-like coupling mechanisms for capacitative calcium entry in corneal endothelial cells. J. Biol. Chem. 277 (19), 16559e16566. Zhang, Y., Li, J., Xie, Q., Bonanno, J.A., 2006. Molecular expression and functional involvement of the bovine calcium-activated chloride channel 1 (bCLCA1) in apical HCO3- permeability of bovine corneal endothelium. Exp. Eye Res. 83 (5), 1215e1224. Zhang, Y., Xie, Q., Sun, X.C., Bonanno, J.A., 2002. Enhancement of HCO 3 e permeability across the apical membrane of bovine corneal endothelium by multiple signaling pathways. Invest. Ophthalmol. Vis. Sci. 43 (4), 1146e1153.
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Experimental Eye Research journal homepage: www.elsevier.com/locate/yexer
Molecular mechanisms underlying the corneal endothelial pump Joseph A. Bonanno* Indiana University, School of Optometry, 800 E Atwater Avenue, Bloomington, IN 47405, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 March 2011 Accepted in revised form 7 June 2011 Available online 15 June 2011
The corneal endothelium is responsible for maintaining the hydration of the cornea. This is through a “Pump-Leak” mechanism where the active transport properties of the endothelium represent the “Pump” and the stromal swelling pressure represents the “Leak”. For the “Pump”, Naþ, Kþ ATPase activity and the presence of HCO3, Cl, and carbonic anhydrase activity are required. Several basolateral (stromal side) anion transporters, apical (facing the aqueous humor) ion channels and water channels have been identified that could support a model for ion secretion as the basis for the endothelial pump, however evidence of sustained anion fluxes, osmotic gradients or the need for water channels is lacking. This has prompted consideration of other models, such as Electro-osmosis, and consideration of metabolite flux as components of the endothelial pump. Although the conditions under which the “Pump” is supported are known, a complete model of the endothelial “Pump” has yet to emerge. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: corneal endothelium transparency ion & fluid transport
1. Introduction The topic of molecular mechanisms underlying the corneal endothelial pump was extensively reviewed in 2003 (Bonanno, 2003). The purpose here is to summarize the important features of the endothelial pump and provide an update on recent significant findings. Development of new techniques, e.g. siRNA knockdown and in vivo viral transfection of shRNA, has produced more specific testing of pump features. Similarly, the use of knockout models (e.g., AQP1) has prompted significant re-thinking of the nature of solutefluid coupling. These molecular approaches together with new studies using customary pharmacological approaches for studying corneal endothelial fluid transport have led to re-evaluations of the corneal endothelial pump. 2. Background The cornea is the major refractive element of the eye. This requires optical transparency and smooth curved surfaces. The cornea has five layers: the outer epithelium, Bowman’s (basement) membrane, the stroma, Descemet’s (basement) membrane, and the inner surface endothelium. The smooth regular surface, tight packing of cells, relative paucity of organelles (especially mitochondria), and lack of blood vessels in the stratified squamous corneal epithelium (w50 mm) reduce light scatter thereby contributing to transparency. The corneal
* Tel.: þ1 812 855 4440; fax: þ1 812 855 7045. E-mail address: [email protected]. 0014-4835/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.exer.2011.06.004
stroma reduces light scatter by its regular spacing among collagen fibers and uniform diameter of fibers. This contrasts with the sclera where spacing and fiber diameters have a very broad distribution. The regular spacing of the stromal fibers is controlled by the hydrophilic glycosaminoglycan (GAGs) ground substance. When the hydration of the stroma is w3.5 mg H2O/mg dry tissue or less, the stroma is relatively transparent. A slit-lamp view of the stroma however, indicates that it scatters more light than the epithelium. This is due to the thin flat keratocytes that lie between stromal lamellae. Keratocytes are responsible for producing collagen and GAGs (among many other extracellular substances) and will be activated during trauma to repair the stroma. Repair alters the expression profile of keratocytes, which can lead to increased light scatter. In contrast, the corneal endothelium is a thin (4 mm) confluent monolayer that has a very high density of mitochondria, but because of its extremely short optical path length, scatters very little light. The epithelium provides the barrier to the outside world, the stroma provides the refractive shape and the endothelium maintains the nutrition of the corneal cells and the hydration of the stroma. Except for oxygen, all the nutrients for the cornea come from the aqueous humor and through the endothelium. For example, glucose transporters are present on both the apical (aqueous humor side) and basolateral (stromal side) endothelial cell membranes to allow transcellular glucose flux (Kumagai et al., 1994). Moreover, 85% of the glucose consumed by the cornea is converted to lactate, which diffuses posteriorly across the endothelium (Riley, 1969). The corneal endothelium also maintains the hydration of the stroma through active transport mechanisms. Because of the swelling pressure (w60 mmHg) exerted by molecular
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repulsion from the highly negatively charged stromal GAGs, bare stroma can swell to many times its normal thickness (see (Kwok and Klyce, 1990) for further details) and as a consequence the spacing between fibers becomes non-uniform, light scatter increases, and corneal transparency is lost. This tendency to swell is counteracted by the endothelial pump through the membrane active transport mechanisms. Steady-state hydration occurs when the endothelial pump rate equals the GAG driven leak. Maurice (Maurice, 1981) called this the “Pump-Leak” mechanism for maintenance of corneal hydration and transparency. Because of the presence of the continuous leak, loss of endothelial ion transport activity leads to corneal edema, loss of transparency, and impaired vision. 3. Corneal endothelial pump description The endothelial pump function is best demonstrated using rabbit corneas mounted in modified Ussing chambers in vitro. If carefully mounted and perfused with appropriate media, the cornea will maintain its thickness for several hours. Another approach is to remove the epithelium, expose the anterior stroma for a short period to Ringer’s solution allowing the stroma to swell, remove the anterior solution and replace it with silicone oil. The corneal thickness will then slowly decrease exponentially in a process called deturgescence. From these experiments it was determined that the cornea will swell, or not deturgesce, if the endothelium is exposed to the cardiac glycoside ouabain, an inhibitor of the Naþ, Kþ ATPase, indicating that the endothelial pump is dependent on primary active transport. Removal of HCO3 from the endothelial perfusing solution had a similar inhibitory effect on the pump and addition of carbonic anhydrase inhibitors slowed the pump by w30%, indicating a major role for HCO3 (Dikstein and Maurice, 1972; Fischbarg and Lim, 1974; Hodson and Miller, 1976; Riley et al., 1995). Because Cl removal did not cause initial swelling, the early pump model was described as an exclusiveHCO3 secretory mechanism. Subsequent studies indicated that Cl was important for maintaining the pump (Winkler et al., 1992), suggesting that an anion exchanger, (Cl/HCO3) played a role. Consistent with an anion transport mechanism, anion transport blockers, e.g. DIDS (4,40 -Diisothiocyano-2,20 -stilbenedisulfonic acid), significantly slow the pump(Kuang et al., 2004a; Riley et al., 1996). There must also be a role for secondary Naþ fluxes because amiloride, which blocks the Naþ/Hþ exchanger and at high concentrations the Naþ channel ENaC (Epithelial Sodium Channel), also slows fluid transport (Liebovitch and Fischbarg, 1982). Assembling these disparate observations into a clear model for endothelial pump function has been very challenging and is described below.
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Moreover, the basolateral membrane HCO3 permeability of the corneal endothelium is significantly higher than the apical permeability (Bonanno et al., 1999) and with the exception of apical anion channels; no apical HCO3 transporter has been identified. Fig. 1 shows a possible bicarbonate transport model for the corneal endothelium. The basolateral Naþ, Kþ ATPase creates a low intracellular [Naþ] and high intracellular [Kþ], and in conjunction with Kþ channels a negative membrane potential of about 55 mV (Watsky and Rae, 1991). On the basolateral side there are two bicarbonate transporters, a 1Naþ:2HCO3 cotransporter (SLC4A4,NBCe1; Sodium Bicarbonate Cotransporter electrogenic) and the Cl/HCO3 exchanger (SLC4A2, AE2; Anion Exchanger),as well as a Naþ/Hþ exchanger (SLC9A6, NHE1; Sodium Hydrogen Exchanger). NBCe1 can directly load HCO3 into the cell. The Naþ/Hþ exchanger can indirectly load HCO3 into the cell because removal of protons favors formation of HCO3 from CO2, catalyzed by carbonic anhydrase II. Conversely, the Cl/HCO3 exchanger is poised to remove HCO3 from the cell and add Cl. Moreover, a basolateral Naþ:Kþ:2Cl cotransporter helps load intracellular chloride to w40 mM, above electrochemical equilibrium (w12 mM). On the apical side, at least two anion channels have been identified: CFTR (Cystic Fibrosis Transmembrane conductance Regulator) and CaCC (Calcium-activated Chloride Channel). Anion permeability of both channels favors Cl vs HCO3 by w4:1.There is no evidence for apical Cl/HCO3 exchange. As such the model (see Fig. 1) predicts that HCO3 is taken up on the basolateral side and efflux of HCO3 across the apical side is through anion channels driven by the negative membrane potential. Fig. 1 shows an additional potential indirect route for apical HCO3 efflux. Because the influx mechanisms for HCO3 exceed direct efflux mechanisms and the differential must be converted to CO2 (Bonanno and Giasson, 1992), the CO2 can diffuse in any direction and some will diffuse across the apical membrane where carbonic anhydrase IV could catalyze the conversion back to
4. The anion (bicarbonate & chloride) secretion model Fluid secretion that is coupled to ion fluxes is dependent on active transport mechanisms to produce local osmotic gradients that move water across the cellular layer. This is best described for secretory glands and kidney reabsorption processes. More recently, evidence for direct coupling of water to ion fluxes in cotransporters has also been presented (Hamann et al., 2003; Meinild et al., 2000) and could have a significant role in fluid absorption across the intestinal mucosa (Loo et al., 1996). The best tissue models for bicarbonate secretion are the gall bladder and pancreas. In both tissues transporters and anion channels have been identified in both basolateral and apical membranes that could provide the net transcellular bicarbonate flux that can unequivocally be measured. In contrast, several groups have measured HCO3 and Cl fluxes across the rabbit corneal endothelium. However, there is no consensus that a net anion flux can be generated (Bonanno, 2003).
Fig. 1. Bicarbonate Secretion Model for Endothelial Pump. Fluid coupled anion secretion requires transendothelial net flux of Cl and/or HCO3. The movement of net negative charge creates a small potential difference (0.5 mV, apical side negative) that attracts Naþ through the paracellular pathway & across the tight junction (TJ). The net flux of NaHCO3 and/or NaCl constitutes the osmotic driving force for water movement. HCO3 uptake on the basolateral membrane is through the actions of the 1Naþ:2HCO3 cotransporter (NBCe1) and the Naþ/Hþ exchanger (NHE1). Cl uptake is primarily via the Naþ:Kþ:2Clcotransporter (NKCC1) and the Cl/HCO3 exchanger (AE2). The high intracellular [Cl] and [HCO3], together with the negative membrane potential can then drive anions across the apical membrane through anion selective channels. An additional route for net HCO3 flux is for the high intracellular [HCO3] to be converted to CO2, facilitated by carbonic anhydrase II (CAII), apical CO2 diffusion and conversion back to HCO3, facilitated by carbonic anhydrase IV (CAIV) at the apical surface. This pathway is less attractive because it does not contribute to the transendothelial potential.
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HCO3 and thereby contribute to a local buildup of osmotic pressure at the apical membrane. The following sections discuss recent studies of several corneal endothelial transport mechanisms and their potential contribution to an anion secretion model. 4.1. NKCC1 The role for Cl is unclear. Intracellular [Cl] is about 40 mM in bovine endothelium (Srinivas et al., 1998), which is above electrochemical equilibrium. This is due to the presence of a bumetanide sensitive basolateral Naþ:Kþ:2Cl cotransporter (NKCC1) (Diecke et al., 1998; Diecke et al., 2005; Jelamskii et al., 2000). Together with the apical Cl channels, the potential for Cl secretion exists. One study could not show any effect of the Naþ:Kþ:2Cl blocker bumetanide on rabbit corneal deturgescence (Riley et al., 1997), however small, but significant effects were noted in a later study (Diecke et al., 2005). Certainly NKCC1 is involved in regulation of cell volume, however given the lack of clear evidence for a net transendothelial Cl flux, the role NKCC1 could have in the endothelial pump is uncertain. One suggestion is that Cl efflux through anion channels offsets the membrane hyperpolarizing effect of the electrogenic1Naþ:2HCO3 cotransporter. By keeping the membrane potential relatively depolarized through Cl efflux, the driving force for continual 1Naþ:2HCO3 influx is maintained. 4.2. NBCe1 The dependence of endothelial fluid transport on active transport and the presence of bicarbonate suggest an important role for the 1Naþ:2HCO3 cotransporter (NBCe1). NBCe1 has two splice variants, pNBCe1 and kNBCe1, for pancreas and kidney type, respectively. The kidney proximal tubule form of NBC (kNBC) has a 1:3 stoichiometry, which favors cellular efflux of HCO3, and the pancreas form of NBC (pNBC) has a 1:2 stoichiometry, which favors HCO3 influx. However, more recent studies have shown that the stoichiometry of either kNBC or pNBC can change depending on the cell type in which it is expressed (Gross et al., 2001). Previous reports indicated the pNBC variant was expressed in bovine (Sun et al., 2000), human (Sun and Bonanno, 2003)and rat corneal endothelium(Bok et al., 2001), and one report suggested both variants were expressed in human endothelium (Usui et al., 1999).Immunohistochemistry studies in cultured and fresh bovine, rat, and human endothelium indicate that NBCe1 exclusively locates to the basolateral membrane; however, one report (Diecke et al., 2004) suggests apical expression as well. NBCe1 specific siRNA knockdown reduced NBCe1 expression by 80e90% four days post transfection in cultured bovine corneal endothelial cells (Li et al., 2005). This treatment significantly reduced basolateral HCO3 permeability and also reduced the basolateral to apical HCO3 flux induced by basolateral to apical [HCO3] gradient. Importantly, apical HCO3 permeability and apical to basolateral HCO3 flux was unaffected by siRNA knockdown. This study shows that functional NBCe1 is only present on the basolateral membrane and that it is necessary for a large portion of basolateral HCO3 influx. 4.3. Calcium-activated chloride channels In bovine corneal endothelial cells, apical HCO3 permeability can be enhanced by increasing [Ca2þ]i via either activation of purinergic receptors (e.g., by ATP) or inhibition of the sarcoendoplasmic reticulum Ca2þ-ATPase (SERCA) (Xie et al., 2002). In either case there is a release of Ca2þ from intracellular stores with concomitant Ca2þ entry from the extracellular bath through storeoperated channels in a process called capacitive calcium entry (CCE). CCE agonists enhanced apical HCO3 permeability in Ca2þrich Ringer solution, but not in the absence of extracellular Ca2þ,
which was blocked by the CCE inhibitor2-aminoethoxydiphenyl borate (2-APB) (Zhang et al., 2006; Zhang et al., 2002). ATP release from endothelial cells does occur and can be stimulated by injury or cell volume increase (Srinivas et al., 2001) indicating that this pathway could be important during cellular stress. A likely candidate for the apical Ca2þsensitive channel is CLCA (chloride channel, calcium-activated) (Cunningham et al., 1995). CLCA1 antiserum detected the 90kD band in corneal endothelium. Immunofluorescence staining showed an apical membrane location (Zhang et al., 2006). In endothelial cells transfected with CLCA1 specific siRNA, CLCA1 expression was reduced by 80%. Activating CCE increased apical HCO3 permeability in siControl transfected cells, while having no effect in CLCA1 specific siRNA transfected cells. Baseline HCO3 permeability however, was not different between control and siRNA treated cells. These studies indicate that the calcium-activated chloride channel (CLCA1) is expressed in corneal endothelial cells and can contribute to Ca2þ dependent apical HCO3 permeability, but not resting permeability across the corneal endothelium. Since release of ATP activates the purinergic pathway, this channel may have a role during stress, e.g. inflammation, to enhance “Pump” activity. 4.4. CFTR Increasing intracellular [cAMP] by activating membrane adenylyl cyclases with forskolin stimulates an NPPB (5-nitro-2-(3phenylpropylamin benzoic acid)) and glibenclamide-inhibitable apical Cl and HCO3 permeability that has been identified as CFTR (Cystic Fibrosis Transmembrane conductance Regulator) in bovine corneal endothelium (Sun and Bonanno, 2002). Knocking down CFTR expression using siRNA eliminated forskolin induced increases in apical Cl and HCO3 permeability (Li et al., 2008). However, the siRNA knockdown had no effect on baseline unstimulated anion permeability. Stimulation of CFTR can also induce a steady-state apical bath alkalinization. In CFTR siRNA treated cells, the baseline bath pH was similar to control, but stimulation did not produce an apical alkalinization. These findings indicate that cAMP induced increases in apical HCO3 permeability in bovine corneal endothelium are due to CFTR. However, CFTR does not have a major role in determining baseline apical chloride or HCO3 permeability. This is consistent with reports indicating that there are no corneal abnormalities in humans with cystic fibrosis and studies showing that a specific CFTR blocker had no significant effect on baseline corneal thickness (Diecke et al., 2004). CFTR may, like CLCA, have a role only during stimulated stressful conditions. 4.5. CA IV As described in Fig. 1, there is the potential for apical CO2 flux contributing to net basolateral to apical HCO3 flux. This requires an apical carbonic anhydrase. An apical CAIV is present in bovine corneal endothelium (Sun et al., 2008). The relatively membrane impermeant carbonic anhydrase inhibitor benzolamide or CAIV siRNA knockdown reduced apical CO2 fluxes due to an imposed [CO2] gradient by w20%, however there was no effect on HCO3 permeability or HCO3 flux (Sun et al., 2008). If a significant net flux of CO2 occurred across the apical membrane with conversion to HCO3 þ Hþ (Fig. 1), then blocking the apical CAIV would cause an alkalinization of the apical bath. However, apically applied benzolamide and siRNA treated cells showed an acidification of the apical side relative to basolateral, which is inconsistent with a net cell to apical CO2 flux. Because CAIV does not facilitate steady-state cell to apical CO2 flux, apical HCO3 permeability, or basolateral to apical HCO3 flux, but apparently maintains apical side pH, CAIV may have a role in buffering the apical surface.
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4.6. Role of NBCe1: in vivo studies
6. Aquaporins
A mouse knockout for NBCe1 exists, however NBCe1 knockouts have significant developmental and systemic abnormalities and die shortly after birth, limiting their usefulness in analyzing the role of NBCe1 in the cornea. Therefore, to follow up on the previous in vitro studies, lentiviral vectors encoding NBCe1 specific shRNA were constructed and injected into the anterior chamber of live rabbits to determine if the bicarbonate fluxes produced by NBCe1 were needed to support corneal endothelial fluid transport in vivo (Liu et al., 2010). This study showed that significant (up to 90%) endothelial NBCe1 knockdown could occur in vivo, however there was large variability in knockdown efficiency. If transendothelial HCO3 transport were an integral part of the endothelial pump, then knocking down NBCe1 should induce significant corneal swelling. Surprisingly, there was no effect on corneal thickness. However, relative to control eyes that were injected with a Lentivirus encoding a scrambled sequence shRNA, the eyes receiving the NBCe1 specific shRNA were more sensitive to the topical carbonic anhydrase inhibitor brinzolamide, i.e. there was more corneal swelling. These results were interpreted to suggest that the corneal endothelium has a large functional reserve (e.g., NBCe1 & CA activity), however they could also be interpreted to suggest that transendothelial HCO3 flux is not an integral part of the “Pump”. In summary, the recent studies presented above do not directly support the anion secretion model.
The role of aquaporins in fluid transport has been controversial. Knockout models of AQP1, the most ubiquitous aquaporin, showed essentially no phenotype unless the animal was stressed by withholding water (Agre, 1998). Subsequent studies indicated that knockout of aquaporins had effects on secretory tissues if the rate of secretion was relatively high (Ma et al., 2000; Ma et al., 1999). Corneal endothelium has a high density of AQP1 located on both apical and basolateral membranes (Kuang et al., 2004b). In the knockout mouse, corneal thickness is thinner than wild type rather than the expected thicker more hydrated cornea if AQP1 was an important part of the “Pump”. The endothelial osmotic permeability is significantly reduced, as expected, and the rate of corneal thickness change in response to an osmotic challenge is slowed. These results suggest that AQP1 is present as a passive water pathway that speeds stromal hydration changes due to osmotic pressure fluctuations. In normal physiological situations this would include eye closure, which swells the cornea by w4%, or exposure to hypo-osmotic conditions, e.g. swimming. This is consistent with the findings in other tissues that aquaporins do not have a role in secretion if the rate is relatively low.
5. The electro-osmosis model The conventional view of epithelial cell secretion and absorption of water is that epithelial cell layers create local osmotic differences in the lateral spaces between the cells and/or on the apical surfaces of cells within an unstirred layer. The osmotic gradients generated serve as the driving forces for water movement across the epithelium. However, in many epithelial cells, including the corneal endothelium, there is no evidence that these gradients exist. This has led to the consideration of other mechanisms, such as ElectroOsmosis (Sanchez et al., 2002). In this process, cells generate a transepithelial potential (0.5 mV apical side negative in corneal endothelium), through the concerted action of apical anion channels (HCO3 and Cl), that draws counter-ions, i.e. Naþ, through a paracellular pathway (the lateral space between cells) lined with fixed negative charges that make the paracellular pathway Naþ specific. This produces electro-osmotic fluid coupling across the tight junction. Evidence for electro-osmosis and against other modes of fluid transport is recently reviewed (Fischbarg, 2010). For electroosmosis, a “leaky” junction with low specific resistance (20 U cm2) must have a relatively intense current density. This high Naþ current generates fluid movement by electro-osmotic coupling. The Naþ current loop is complete due to the presence of apical Naþ channels (ENaC) (Mirshahi et al., 1999). Of interest, apical membrane density of ENaC can be enhanced by SGK1 activity (serum glucocorticoid kinase), which has also been shown to increase Na/K-ATPase activity in corneal endothelium (Hatou et al., 2009; Rauz et al., 2003). This could be interpreted in favor of electro-osmosis or the increased ENaC density may simply be needed to offset the hyperpolarizing effect of increased Na/K-ATPase activity in order to maintain a stable membrane potential. Moreover, glucocorticoids thin rabbit (Hara, 1970), but not human corneas (Baum and Levene, 1968). A major problem with electro-osmosis in corneal endothelium is that a significant reduction in endothelial cell density, which is a normal process of ageing, does not result in reductions in endothelial function or production of corneal edema. The integrated effect of the high current density would be dissipated since the total paracellular area is significantly decreasing with age.
7. Fluid transport agonists The most highly studied fluid transport agonists in corneal endothelium are those that result in increased [cAMP]. Adenosine was the first agonist discovered (Dikstein and Maurice, 1972) and was subsequently shown that it increased [cAMP] in endothelial cells (Riley et al., 1996) through activation of A2b receptors (TanAllen et al., 2005). Other agonists that increase [cAMP], e.g. forskolin or the phosphodiester inhibitor rolipram, also cause corneal thinning (Wigham et al., 2000). It is important to note that this could occur in de-epithelialized preparations so it was not due to the well-characterized corneal epithelial cAMP dependent Cl secretion (Klyce, 1975). Besides being protective for corneal endothelial cells (Li et al., 2011), increased cAMP will phosphorylate and activate the apical CFTR channel leading to increased Cl and HCO3 flux (Sun et al., 2003). It also relaxes endothelial cells leading to better cellecell apposition and increased transendothelial resistance (Ramachandran and Srinivas, 2010). ATP is also a potential fluid transport agonist. Purinergic receptor activation increased apical anion channel permeability as described above. Moreover, ATP released by the endothelial cells, can be broken down to adenosine by the presence of eco ATPases (Soltau et al., 1993). Again, the presence of this physiologic response is probably in place to stimulate endothelial function during inflammatory, oxidative, or traumatic stress to the cell layer, but this will need to be tested. 8. Facilitated lactate transport Often overlooked is the fact that in the in vivo cornea there are substantial bulk gradients of ions, e.g. glucose, HCO3 and lactate (Chhabra et al., 2009). [HCO3] is higher in the anterior chamber than in the cornea, especially when the eyelid is open. Thus HCO3 will diffuse into the cornea passively in the opposite direction of the putative bicarbonate secretory mechanism. The cornea gets all of its glucose from the anterior chamber. Glucose in the cornea is consumed thereby maintaining an inward gradient for glucose diffusion. The high concentration of mitochondria in the endothelial cells would minimize glucose consumption allowing for supply to the very glycolytic keratocytes and epithelial cells. Overall, Riley (1969) has shown that 85% of glucose consumed in the cornea is converted to lactic acid. Therefore, for every 100 glucose molecules
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channel activation and greater barrier function. Further studies are needed to determine if this mechanism could be used as a therapy for corneal edema. References
Fig. 2. Facilitated Lactate Transport Model. The bulk movement of lactic acid from cornea to anterior chamber via transcellular Monocarboxylate Cotransporters (MCTs) could contribute to net fluid movement out of the cornea. Intracellular buffering by the high intracellular [HCO3], provided by the 1Naþ:2HCO3 (NBCe1), Naþ/Hþ exchanger (NHE1), and carbonic anhydrase II work to facilitate basolateral lactate:Hþ entry. Apical efflux through an MCT is facilitated by robust apical surface buffering, which is supplied by aqueous humor HCO3 and apical surface carbonic anhydrase IV.
entering the cornea, 170 lactate molecules are produced. Klyce (1981) demonstrated that the epithelium is impermeable to lactate and that the [lactate] in the cornea was about twice that in the aqueous humor, so there is a large gradient for lactate flux from cornea (w13 mM) to anterior chamber (w7 mM) across the endothelium. Lactate:Hþ cotransport was demonstrated in the endothelium (Giasson and Bonanno, 1994) and the monocarboxylate transporters (MCTs) responsible are being studied in our laboratory. Corneal hypoxia increases [lactate] and causes corneal swelling (Klyce, 1981) indicating that lactate is an important osmolyte. It follows that any process that facilitates lactate efflux from the cornea will help maintain dehydration. Conversely, any interference with lactate efflux will cause corneal swelling. Fig. 2 illustrates a model for lactate efflux across the endothelium that is facilitated by HCO3 buffering, carbonic anhydrase activity, 1Naþ:2HCO3 cotransport, and Naþ/Hþ exchange. An important role for buffering acid is supported by studies indicating that deturgescence can be maintained in the absence of HCO3 if a high concentration of other buffers are present (Doughty and Maurice, 1988). Studies in our laboratory are currently examining the extent to which this process may contribute to the endothelial pump. 9. Conclusion The corneal endothelial pump is an active transport dependent process that requires both Cl and HCO3, but is highly dependent on HCO3 and facilitated by carbonic anhydrase activity. While a HCO3 secretory model for the pump has been preferred, there are several issues that argue against this interpretation. These include: the lack of apical anion transporters, equivocal measures of net anion fluxes from stroma to anterior chamber (probably due to the extremely leaky nature of the endothelium), no data indicating the presence of local osmotic gradients, a bulk fluid HCO3 gradient that is against the putative secretory mechanism, and the greater potential for a Cl secretory mechanism. Alternatives such as electro-osmosis are being considered, but this suffers from the fact that significant reductions in electro-osmotic potential, i.e. the paracellular space, do not have direct effects on fluid pumping. The lactate efflux mechanism is attractive, but requires significant experimental testing. Increasing endothelial cAMP enhances pump function by a combination of anion
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